Zyxin is important for the stability and function of podocytes, especially during mechanical stretch

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch.

(a) Mechanically stretched primary podocytes showed an increased zyxin (magenta) localization from focal contacts to actin filaments (white).Primary podocytes showed no zyxin accumulation in the nucleus after mechanical stretch.Synaptopodin (green) was used as a podocyte control.The scale bar represents 25 μm.(b) Immunostaining of vinculin (magenta) and F-actin (green) in Ctrl and Zyx-KO mechanically stretched immortalized podocytes.For quantification vinculin-positive signals were segmented and quantified (Fig. 2h).The scale bar represents 100 μm.(c) Migration assays were performed to study the migration of Zyx KO podocytes.For this, Zyx KO and Ctrl podocytes were cultured in a migration chamber as confluent layers which were separated into two distinct areas by a removable plastic partition in the middle of the chamber.After removing the partition, the dynamics of podocytes were followed over 22 hours by light microscopy.Images show different time points (0 and 22 hours).The distance between the migrating areas is marked by black (dashed) lines and revealed that Zyx KO podocytes migrated significantly faster into the gap than the control podocytes.Data are presented the mean values of individual field of views (n=21; **** p<0.0001).

Supplementary Table 2:
LC-MS/MS parameter (data independent mode; quantitative data)

Data independent analyses reversed phase liquid chromatography
Ultimate 3000 RSLC (Thermo Scientific)

Fig
Fig. S1: Additional information for figures 1, 2 and 4. (a) Mechanically stretched primary podocytes showed an increased zyxin (magenta) localization from focal contacts to actin filaments (white).Primary podocytes showed no zyxin accumulation in the nucleus after mechanical stretch.Synaptopodin (green) was used as a podocyte control.The scale bar represents 25 μm.(b) Immunostaining of vinculin (magenta) and F-actin (green) in Ctrl and Zyx-KO mechanically stretched immortalized podocytes.For quantification vinculin-positive signals were segmented and quantified (Fig. 2h).The scale bar represents 100 μm.(c) Migration assays were performed to study the migration of Zyx KO podocytes.For this, Zyx KO and Ctrl podocytes were cultured in a migration chamber as confluent layers which were separated into two distinct areas by a removable plastic partition in the middle of the chamber.After removing the partition, the dynamics of podocytes were followed over 22 hours by light microscopy.Images show different time points (0 and 22 hours).The distance between the migrating areas is marked by black (dashed) lines and revealed that Zyx KO podocytes migrated significantly faster into the gap than the control podocytes.Data are presented the mean values of individual field of views (n=21; **** p<0.0001).

Fig. S2 :
Fig. S2: Ingenuity Pathway Analysis (IPA).IPA was used to identify zyxin containing protein-protein interaction networks.(a) Organic representation of the interaction network.The organic layout style is based on the force-directed layout paradigm.(b) Subcellular illustration places the nodes into a simplified view of subcellular compartments (c) Hierarchical representation of the interaction network highlights the main flow within a directed graph.(d) Prediction Legend: Red: up-regulated; green: down-regulated in Zyx-KO podocytes compared to controls (measured and quantified by LC-MS/MS).Orange: predicted up-regulation; blue: predicted down-regulation.

Fig. S3 :
Fig. S3: Kidney expression pattern of Sirpa and Iqgap2.(a) Database analysis using the Kidney Cell Explorer by Ransick et al. based on a single-cell RNA sequencing data set of murine kidneys showed the expression pattern for all kidney cell fractions [PMID: 31689386].Colour code: Red means a high average expression; Blue: low expression.(b) Legend of the numerical labelling of Fig. S3a.Data and illustrations are from KidneyCellExplorer (https://cello.shinyapps.io/kidneycellexplorer/;Ransick et al., 2019).

Fig. S4 :
Fig. S4: Correlation of zyxin expression with proteinuria (Minimal Change Disease Samples).Zyxin expression is not correlated with urine protein-creatine ratio (a) and proteinuria (b) in different MCD patient cohorts.(a) The data were analyzed using an Affymetrix Human Gene 2.1 ST Array [PMID: 26150607].(b) Data were analyzed by RNA_Seq [PMID: 36442540].Both data sets were taken from Nephroseq Research Edition (Ann Arbor, University of Michigan).